2.1 CURRENT METHODS FOR INTRODUCING POLYNUCLEOTIDES INTO CELLS
Contemporary science has set, amongst its goals, the sequencing of the entire human genome, with the intention of using the genetic information obtained to further our understanding of physiology and to expand the opportunities for treatment of disease. One avenue of treatment currently being explored is the technique of gene therapy, in which a polynucleotide is introduced into a cell to confer a therapeutic benefit, for example, to provide a functional copy of a gene where the intrinsic gene is defective.
A number of methods have been conventionally used to introduce polynucleotides into cells, including calcium phosphate-DNA precipitation, clectroporation, DEAE-dextran transfection, microinjection, and the use of a "gene gun" (see Australian Patent No. 9068389). Inefficiency of incorporation of polynucleotide and/or technical obstacles render each of these techniques problematic.
In hopes of improving polynucleotide delivery, viral vectors, including retrovirus, adenovirus, and adeno-associated virus vectors have been developed. Each of these three types of viral vectors, however, has serious disadvantages. Retroviruses insert polynucleotides only into cells which are actively dividing, and may act as insertional mutagens; adenovirus vectors provoke an immune response which eliminates vector from the host, thereby rendering any therapeutic benefit transient; and adeno-associated virus vectors are difficult to produce in large quantities (Marshall, 1995, Science 269:1050-1055; Paillard, 1998, Human Gene Therapy 9:1699-1700).
In order to avoid the disadvantages associated with viral vectors and conventional transfection methods, a number of alternative means and compositions for introducing nucleic acid into a cell have been devised. Such alternatives include lyophilized formulations of polynucleotide-lipid complexes (see, for example, International Patent Application Nos. PCT96US7867 and PCT 96US7866); polynucleotides linked to a dendrimer polycation (to improve transfection efficiency; U.S. Pat. No. 5,661,025); polynuclcotide compositions comprising a membrane-permeabilizing agent to transport the polynucleotide across the target cell membrane (International Patent Application No. PCT 93US3406); the use of polylysine as a DNA condensing agent (Wadhwa et al., 1995, Bioconjugate Chemistry 6:283-291), optionally linked to a carrier protein such as transferrin; and DNA trapped in liposomes (Ledley et al., 1987, J. Pediatrics 110:1) or in proteoliposomes (Nicolau et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1068). Receptor-mediated gene transfer techniques have been developed which rely on specific receptor/ligand interactions (Wu et al., 1988, J. Biol. Chem. 263:14621-14624; Christiano et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:2122-2126; Huckett et al., 1990, Biochem. Pharmacol. 40:253-263; Perales et al., 1994, Eur. J. Biochem. 226:255-266). U.S. Pat. No. 5,589,466 by Felgner provides for the introduction of DNA into interstitial spaces, where it becomes available for cellular uptake. Truong-Le et al., 1998, Human Gene Therapy 9:1709-1717 report controlled gene delivery by DNA-gelatin microspheres having a size range of 200-700 nm, wherein transfection of cells was enhanced by incorporating chloroquine, which interferes with endosome acidification, into the microspheres.
It remains to be seen whether such methods and compositions will be successful in avoiding polynucleotide degradation, gaining access to the cytosol, and achieving sufficient proximity to the cell nucleus to result in efficient nuclear uptake.
2.2. CLATHRIN-COATED VESICLE MEDIATED UPTAKE AND TRANSPORT
In nature, there are two main mechanisms for internalization of substances: phagocytosis (literally, "cell eating"), in which relatively large particles (&gt;0.5 .mu.m diameter) are ingested, and pinocytosis ("cell drinking"), whereby smaller particles are internalized into small (&lt;0.2 .mu.m diameter) vesicles (reviewed in Mellman, 1996, Annu. Rev. Cell Dev. Biol. 12:575-625). Uptake by pinocytosis typically occurs at a specialized site in the plasma membrane referred to as a clathrin-coated pit (Id.).
Uptake of extracellular fluid and receptor-bound ligands is now known to occur via clathrin-coated pits, which serve as portals of entry into an intracellular system of vesicular organelles referred to as the "endocytic pathway". The endocytic pathway serves not only as a mechanism by which compounds in the extracellular space may enter cells, but also as a means for receptor recycling, as once a receptor releases its ligand in an internal vesicle, it may be returned to the cell surface via the pathway. Individual receptors have been estimated to be recycled at a rate of ten times per hour (Steinman et al., 1983, J. Cell. Biol. 96:1-27). This indicates that clathrin-mediated uptake and the endocytic pathway play a substantial and dynamic role in cell metabolism. Indeed, it has been reported that cells such as macrophages and fibroblasts internalize more than 200 percent of their entire surface area every hour (Id.).
A clathrin-coated pit closes around material to be internalized (referred to herein as its "cargo") by a series of molecular interactions and structural changes (discussed in greater detail in the next subsection), which result in a scaled, cargo-carrying clathrin-coated vesicle budding off the plasma membrane and entering the cytoplasm. Shortly after being internalized in this manner, the vesicle loses its clathrin coating and fuses with an early endosome (Helenius et al., 1983, Trends Biochem. Sci. 8:245-250). The primary role of early endosomes is to sort the "cargo" into molecules which are to be recycled (e.g. receptors which are to be sent back to the cell surface) or metabolized (in a lysosome; Mellman, 1996, Annu. Rev. Cell Dev. Biol. 12:575-625). Vesicles containing molecules which are to be metabolized are transferred to the perinuclear cytoplasm, where they fuse with late endosomes. The late endosomes, in turn, fuse with lysosomes, where the cargo is enzymatically digested.
The pathway from clathrin-coated vesicles to early endosomes, late endosomes and finally lysosomes is marked by a decrease in intra-vesicular pH (Mellman, 1992, J. Exp. Biol. 172:39-45). This decrease in pH is mediated by a V-ATPase, which promotes proton conduction across the vesicle membrane (Forgac, 1992, J. Exp. Biol. 172:155-169; Nelson, 1992, J. Exp. Biol. 172:149-153).
In early endosomes, the pH is mildly acidic (5.5-6.3), favoring release of receptor-bound ligand, but not sufficient to damage the receptor, thereby enabling recycling (Mellman, 1992, J. Exp. Biol. 172:39-45). It has been observed that many ligands dissociate from their receptors at pH less than 7 (Maxfield and Yamashiro, 1987, Adv. Exp. Med. Biol. 225:189-198). At pH less than 6, iron is released from differic transferrin (IdI., Princioto and Zapolski, 1975, Nature 255:87; Aisen and Listowsky, 1980, Annu. Rev. Biochem. 49:367), and the epidermal growth factor receptor undergoes a large scale conformational change which releases its ligand (Maxfield and Yamashiro, supra, DiPaola and Maxfield, 1984, J. Biol. Chem. 259:9163).
Progrcssing along the endocytic pathway, the intravesicular environment becomes more favorable to cargo degradation. The pH of late endosomes is generally less than 5.5, and the lysosomal pH may be as low as 4.6 (Mellmaii, 1992, J. Exp. Biol. 172:39-45; Komfeld and Mellman, 1989, Annu. Rev. Cell Biol. 5:483-525). Moreover, the content of acid hydrolases increases (Id.). Sometimes the cargo being degraded is a receptor; one mechanism of receptor down-regulation is the destruction of ligand-receptor complexes in lysosomes (as observed for the Fe receptor bound to IgG; Mcllman and Plutner, 1984, J.Cell. Biol. 98:1170-1176; Mellman and Ukkonen, 1984, J. Cell Biol. 98:1163-1169; Ukonnen et al., 1986, J. Exp. Med. 163:952-971).
In addition to its role in uptake of molecules and receptor recycling, the endocytic pathway is exploited by certain pathogenic agents, including diphtheria toxin and various viruses, such as influenza virus and Semliki Forest virus (Maxfield and Yamashiro, 1987, Adv. Exp. Med. Biol. 225:189-198; Olsnes and Sandvig, 1983, in "Receptor-inediated Endocytosis: Receptors and Recognition", Cuatrecasa and Roth, eds., Chapman & Hall, London, pp. 187-236; Klielian and Helcnius, 1985, J. Cell Biol. 101:2284; White et al., 1980, J. Cell Biol. 87:264; Marsh et al., 1983, Cell 32:931). At acidic pH, the toxin or viral coat protein, as the case may be, undergoes a conformational change which permits its passage out of the vesicle and into the cytosol.
Entry into and passage through the endocytic pathway is a rapid process which involves a significant portion of the intracellular volume. By following the progress of the fluid phase marker horseradish peroxidase ("HRP"), it was found that, for the first two minutes after exposure of a baby hamster kidney cell to HRP, the volume density of labeled structures increased rapidly, and then remained constant for the next 13-18 minites at a plateau level accounting for approximately 0.65 percent of the cytoplasmic volume (Griffiths et al., 1989, J. Cell Biol. 109 :2703-2720). Subsequently, the volume density again increased rapidly (as HRP reached the lysosomal compartment), to reach a second plateau between 30 and 60 minutes of labeling, to constitute 3.5 percent of the cytoplasmic volume (Id.).
2.3. MOLECULAR ASPECTS OF CLATHRIN-COATED VESICLE FORMATION
A number of molecules participate in the formation of clathrin-coated vesicles from the plasma membrane, including, in addition to clathrin, AP2 (for "adaptor protein" or "assembly protein") and dynamin (reviewed in Schmid, 1997, Annu. Rev. Biochem. 66:511-548). Upon binding, AP2 triggers assembly of a clathrin lattice (Id.). Dynamin, a GTPase, promulgates structural changes which result in the sealing and budding of the clathrin-coated vesicle from the plasma membrane (Id.).
Clathrin is a protein complex consisting of three 192 kDa heavy chains, each bound to either of two different light chains having molecular weights of approximately 30 kDa. The complex is referred to as a "triskelion" because it has a three-legged appearance (Kirchhausen et al., 1986, J. Ultrastructur. Mol. Struct. Res. 94:199-208; Ungewickelland and Branton, 1981, Nature 289:420-422). In solution, clathrin triskelions self-assemble to form closed polyhedral structures called "cages" (Crowther et al., 1976, J. Mol. Biol. 103:785-798; Woodward, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:4394-4398). In the plasma membrane, polyhedral assemblies of clathrin form coated pits which increase in curvature as cargo from the extracellular environment is engulfed; it is believed that the shape changes in the clathrin pit rcquired for invagination involve the incorporation of pentagonal arrangements of triskelions in an otherwise hexagonal array (Schmid, 1997, Annu, Rev. Biochem. 66:511-548).
AP2 plays a critical role in the attachment of clathrin to membranes, being first recruited to the membrane surface in order to provide a binding site for clathrin (Mellman, 1996, Annu. Rev. Cell Dev. Biol. 12:575-625; Chang et al., 1993, EMBO J. 12:2169-2180; Peeler et al., 1993, J. Cell. Biol. 120:47-54; Robinson, 1994, Curr. Opin. Cell Biol. 6:538-544; Traub et al., 1995, J. Biol. Chem. 270:4933-4942). Moreover, AP2 recruits membrane proteins for uptake by means of a diversity of molecular localization signals comprised in molecules (such as receptors) destined to be clathrin-coated vesicle cargo; a number of such signals have been characterized (Mellman, 1996, Annu. Rev. Cell Dev. Biol. 12:575-625). Broadly defined peptide consensus sequences may include aromatic (usually tyrosine) residues in proximity to one or more amino acids with large hydrophobic side chains (Trowbridge et al., 1993, Annu. Rev. Cell Biol. 9:129-161), or vicinal leucine residues (or leucine and another small hydrophobic amino acid), the latter being favored among receptors expressed in leukocytes (Matter et al., 1994, J. Cell. Biol. 126:991-1004; Hunziker and Fumey, 1994, EMBO J. 13:2963-2967).
Although its role is not yet completely understood, dynamin, in its GDP-bound state, is believed to interact with the clathrin lattice of an invaginated coated pit, whereupon binding of GTP triggers redistribution of the dynamin to form a constricted neck at the plasma membrane, thereby initiating the budding process. GTP hydrolysis then drives a tightening of the neck, resulting in the detachment of a sealed clathrin-coated vesicle into the cytoplasm. The diameter of the clathrin-coated vesicle is approximately 80-100 nm (.08-0.1 .mu.m; Griffiths et al., 1989, J. Cell Biol. 109:2703-2720).
2.4. pH SENSITIVE HYDROGELS A hydrogel is a three-dimensional polymeric network (i.e., matrix) containing a substantial (usually greater than 20 percent) amount of water (Br.o slashed.ndsted and Kopecek, 1992, in "Polyelectrolyte Gels", American Chemical Society, Washington, D.C., pp. 285-304; Wichterle and Lim, 1960, Nature 185:117). Because of their high water content, hydrogels tend to be compatible with biological systems and therefore have been used in various medical applications, including tissue implants, soft contact lenses, and drug delivery systems (Mack et al., 1987, in "Hydrogels in Medicine and Pharmacy", Peppas, ed., CRC Press, Boca Raton, Fla., vol. III, pp. 65-93).
Polymeric hydrogels can exist in two distinct phases: collapsed (also referred to as contracted, condensed, or compressed) and expanded (also referred to as swollen or decompressed). Volume transition occurs between these phases either continuously (gradually) or discontinuously (abruptly), depending on the nature of the polymer and a variety of other factors, including temperature, solvent composition, pH, ionic composition, electric field, and light. When a hydrogel expands, it absorbs water.
Phase transitions in gels have been of particular interest recently, and gels have been developed which expand or contract as necessary to serve a variety of purposes, including providing a superabsorbent diaper, facilitating drug delivery, and mimicking muscle contraction (see, for example, Tanaka, 1992, in "Polyelectrolyte Gels", American Chemical Society, Washington, D.C., pp. 1-21). Discontinuous phase transition may potentially result in a thousand-fold or greater change in gel volume.
Discontinuous phase transition results from an imbalance between attractive (collapsing) and repulsive (expanding) interactions between the polymer constituents of a gel. Such interactions may be ionic, hydrophilic/hydrophobic, van der Waals, or hydrogen-bonding in character (Id.). Electrostatic (ionic) interactions between moieties in the polymer may act as particularly powerful forces to expand or collapse a gel. For example, if a gel having uncharged moieties (e.g., --NH.sub.2) is placed in conditions (e.g., acidic pH) which render its moieties similarly charged (e.g., --NH.sub.3.sup.+), the consequent electrostatic repulsive interaction will act like an internal pressure, and the gel will expand rapidly in order to increase the distance between the like charges. Hydrogols that undergo phase transition in response to changes in pH typically contain pendant acidic or basic groups, such as carboxylic acids and primary amines, or strong acids and bases, such as sulfonic acids and quateniary ammonium salts, which change ionization state as pH increases or decreases (Br.o slashed.ndsted and Kopecek, 1992, in "Polyelectrolyte Gels", American Chemical Society, Washington, D.C., pp. 285-304; Kopecek et al., 1971, J. Polym. Sci. 9:2801).
When used in the context of drug delivery, hydrogels are believed to permit the diffusion of drugs in and out of the gel by, in the case of hydrophilic low molecular weight drugs, a "pore mechanism", and, for more hydrophobic compounds, a "partition mechanism", The permeability of various compounds, up to a molecular weight of 70,000 Da, through lyophilized polyacrylonitrile was observed (Dabrovska et al., 1978, J. Biomed. Mater. Res. 12:591). The extent of cross-linking has been found to be important for pH-dependent permeability (Weiss et al., 1986, AIChE Symposium Series 82:85). It has also been observed that by adding hydrophobic groups to a pH-sensitive hydrogel, the pH change required for phase transition may be increased (by stabilization of the collapsed phase; Philippova et al., 1997, Macromolecules 30:8278-8285).
Recently, Kiser et al. (1998, Nature 394:459-462) produced a synthetic mimic of a naturally occun-ing secretory granule in the form of a microsphere fabricated from a pH and ion-sensitive polymer containing the anti-cancer drug doxorubicin hydrochloride, protecting the microspheres from pH changes with a lipid bilayer coating (U.S. Pat. No. 5,654,006 by Fernandez and Knudson). The lipid bilayer could be breached to result in drug release by placing the particle in an electroporation field (see also Siegel, 1998, Nature 394:427-428).